Transformed cell, method of screening anti-aging agent and anti-aging agent

ABSTRACT

This invention provides a novel transformed cell useful in constructing an anti-aging agent screening system, a screening method which uses the same and an anti-aging agent, and it relates to a transformed cell in which a gene coding for (a) a protein capable of phosphorylating p38 protein, or (b) p38 protein, a mutant of p38 protein, a kinase domain of p38 protein, a kinase domain of p38 protein mutant or a fusion protein containing them is transformed into a normal cell, a screening method which uses this transformed cell and an anti-aging agent which uses a compound obtained by the screening method as the active ingredient.

This application is a National Phase entry of PCT/JP02/07182, filed Jul.15, 2002, which claims priority to Japanese Application Numbers2001-286412 filed Sep. 20, 2001 and 2001-215576 filed Jul. 16, 2001,respectively.

TECHNICAL FIELD

This invention relates to a novel transformed cell, a method forscreening an anti-aging agent and an anti-aging agent.

BACKGROUND OF THE INVENTION

Cellular senescence is conditions under which a cell that shouldoriginally carry out cell growth cannot perform cell division inresponse to growth stimulation. A senescent cell is characterized by itslarge flat nucleus and a cellular senescence-specific β-galactosidase(senescence-associated β-galactosidase; SA-β-galactosidase) activity. Inaddition to morphological changes, the cellular senescent cell(senescent cell) shows characteristic changes in the gene expressionpatterns and the like.

There are many causes which trigger cellular senescence. For example,human normal fibroblast is a cell most generally used in the studies oncellular senescence. It is known that cellular senescence is inducedwhen this is exposed to ionizing radiation or cultured under a highoxygen partial pressure. Also, in recent years, it has been reportedthat cellular senescence is rapidly induced after about 1 week when anactivated oncognic RAS is expressed by force in human normal fibroblast.

However, the cellular senescence most thoroughly studied is a cellularsenescence by the replicative life span (telomere-dependent) in which anormal cell reaches an aging cell after a finite number of celldivisions.

Since the cellular senescence generated by these different causesfinally shows the same phenotype, it is considered that it occurs by thesame molecular mechanism, but its details are not clear.

The cellular senescence based on the replicative life span is aphenomenon in which a normal cell becomes senescent cell after a certainnumber of cell divisions which vary depending on the kind of the cell.In the case of the most frequently used human normal fibroblast, a fetalcell shows an aging phenotype after about 60 to 80 times of celldivision. This phenomenon was reported by Hayflick in 1962. However, ithas been unclear for a long time that intracellular mechanism of therecording of the number of cell divisions. In recent years, it has beenshown that DNA replication of the chromosomal terminal telomere is notcomplete due to the so-called “end replication problem”, and thetelomere length is shortened from about 50 to 200 base pairs per celldivision, and that a cellular senescence is induced when this shorteningreaches its threshold value. However, it is not clear that in whatmanner the cellular senescence occurs by the shortening of telomerelength and whether or not its molecular mechanism is identical tocellular senescence caused by other causes.

On the other hand, MAPK (Mitogen-activated protein kinase) pathway is acascade of protein kinases which exist in the cytoplasm and areactivated by the intracellular and extracellular stimuli and therebyphosphorylate and activate downstream inactive protein kinases. Thefinal target kinase MAPK phosphorylates and activates a specifictranscription factor, and the cell responds to stimuli through theinduction of the expression of a group of genes by the transcriptionfactor.

The upstream kinase which phosphorylates the final target kinase MAPK iscalled MAPKK (MAPK2K), and the upstream kinase which phosphorylates theaforementioned MAPKK is called MAPKKK (MAPK3K). Two or more of the MAPKpathway are present, and MAPK, MAPKK and MAPKK are known as each ofthem.

The classical MAPK pathway which was discovered and analyzed earliest inthe history comprises Raf-Mek-Erk, and it responds to mainlyextracellular stimuli, such as growth factor and the like and isactivated via Ras. PD98059 is known as a specific inhibitor of Mek.

On the other hand, JNK (c-Jun N-terminal kinase) and p38 protein areknown as the MAPK which is induced by cytokine of tumor necrosis factor(TNF) and the like that induce stress and apoptosis. Also, the MAPKK andMAPKKK of JNK and p38 protein are not single, but two or more of themare respectively known.

SB203580 is known as a compound which specifically inhibits the functionof the p38 protein, and it is known that this compound inhibitsproduction of inflammatory cytokines interleukin-1, interleukin-6,interleukin-8 or TNF and therefore is useful a therapeutic agent ofinflammatory diseases (WO 97/33883).

DISCLOSURE OF THE INVENTION

Inventors of the present invention have conducted intensive studies onthe cause of generating cellular senescence and found that the p38 MAPKpathway takes a direct role in the cellular senescence induced by two ormore causes. Though the p38 MAPK pathway has been known as a MAPKpathway which induces stress or apoptosis as described in the above, itsrelationship with cellular senescence has been completely unknown.

In addition, the inventors of the present invention have also foundbased on this finding that when a gene coding for a protein which canactivate p38 protein by its phosphorylation is introduced into a youngcell, cellular senescence can be rapidly induced in the aforementionedjuvenile cell.

The novel senescent cell obtained in this manner can be prepared withina short period of time in comparison with a natural senescent cellobtained via scores of times of natural division, and is markedly usefulin constructing a screening system for anti-aging agents.

In addition, the inventors of the present invention have also and newlyfound that a known inhibitor for p38 protein shows anti-aging activityupon the aforementioned novel senescent cell and natural senescent cell.The invention is based on these findings.

An object of the invention is to provide a novel transformed cell usefulin constructing a screening system for anti-aging agents, a screeningsystem capable of using the same and an anti-aging agent.

More specifically, an object of the invention is to provide a noveltransformed cell in which a gene coding for

(a) a protein capable of phosphorylating p38 protein, or

(b) p38 protein, a mutant of p38 protein, a kinase domain of p38protein, a kinase domain of p38 protein mutant or a fusion proteincontaining them is transformed into a normal cell.

Also, another object of the invention is to provide a method forscreening a compound, which comprises

(1) a step for allowing a normal senescent cell or a normal cellintroduced with a gene capable of inducing cellular senescence tocontact with a substance to be tested, and

(2) a step for analyzing an aging index in the cell.

In addition, still another object of the invention is to provide ananti-aging agent which comprises a compound obtained by the abovescreening method and an anti-aging agent which comprises a p38 proteininhibitor, as an active ingredients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph which is a substitute for a drawing, showingthe presence or absence of β-galactosidase activity in a juvenile celland a senescent cell of human normal fibroblast WI-38.

FIG. 2 is a photograph which is a substitute for a drawing, showing aresult of western blotting of each of the cell lysates shown in FIG. 1using an anti-p38 antibody.

FIG. 3 is a graph showing cell growth curves of a juvenile cell and asenescent cell of human normal fibroblast WI-38 in the presence orabsence of a p38 protein inhibitor, SB203580.

FIG. 4 is a graph showing the ratio of S phase cell in a juvenile celland a senescent cell of human normal fibroblast WI-38 in the presence orabsence of a p38 protein inhibitor, SB203580.

FIG. 5 is a graph showing cell growth curve of the transformed cell ofthe present invention in the absence [graph (a)] or presence [graph (b)]of a p38 protein inhibitor, SB203580.

FIG. 6 is a graph showing the ratio of S phase cell in the transformedcell of the present invention, by FACS analysis.

FIG. 7 is a photomicrograph which is a substitute for a drawing, showingthe presence or absence of β-galactosidase activity in the transformedcell of the present invention.

FIG. 8 is a graph showing a result of the determination ofβ-galactosidase activity in the transformed cell of the presentinvention.

FIG. 9 is a photograph which is substitute for a drawing, showing aresult of western blotting of the transformed cell of the presentinvention.

FIG. 10 is a photomicrograph which is a substitute for a drawing,showing morphological changes of the transformed cell of the presentinvention by varied concentration of added tamoxifen.

FIG. 11 is a graph showing variation per day in the growth ratio of thetransformed cell of the present invention by varied concentration ofadded tamoxifen.

FIG. 12 is a graph showing variation per day in the growth ratio of thetransformed cell of the present invention in the presence or absence ofadded tamoxifen and PD98059.

FIG. 13 is a graph showing a result of the quantitation ofβ-galactosidase in the transformed cell of the present invention in thepresence or absence of added tamoxifen and PD98059.

FIG. 14 is a graph showing variation per day in the growth ratio of thetransformed cell of the present invention in the presence or absence ofadded tamoxifen and SB203580.

FIG. 15 is a graph showing a result of the quantitation ofβ-galactosidase in the transformed cell of the present invention in thepresence or absence of added tamoxifen and SB203580.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is described below in detail.

The transformed cell of the present invention can be obtained byintroducing a gene coding for (a) a protein capable of phosphorylatingp38 protein, or (b) p38 protein, a mutant of p38 protein, a kinasedomain of p38 protein, a kinase domain of p38 protein mutant, or afusion protein containing p38 protein, a mutant of p38 protein, a kinasedomain of p38 protein or a kinase domain of p38 protein mutant, into anormal cell for transformation.

The term “p38 protein” as used herein means an enzyme of a monomercomprising a catalytic subunit of about 38,000 in molecular weight,which is one of the serine/threonine kinases and which belongs to theMAP kinase super family carrying out intracellular signal transduction.The host cell which can be used for preparing the transformed cell ofthe present invention, as well as the kinds of animal, and tissue fromwhich it is derived and the number of cell divisions (namely, whether itis a young cell or senescent cell), is not particularly limited with theproviso that it is a cell which expresses p38 and from which cellularsenescence can be induced by introducing a specified gene that can beused in the present invention (by further allowing an appropriate ligandto perform the reaction, if necessary).

As the host cell, for example, a mammal-derived cell is desirable, and amammal-derived normal cell is more desirable. Examples of themammal-derived normal cell include normal cells (e.g., a normalfibroblast, a normal keratinocyte, a normal mammary gland epithelialcell, a normal vascular endothelial cell and the like) derived fromhuman, monkey, mouse, rat or hamster. Also as the aforementioned hostcell, it is desirable to use a human-derived normal cell, and it is moredesirable to use a human normal fibroblast. Examples of the human normalfibroblast include, WI-38 (ATCC, CCL-75), MRC-5 (ATCC, CCL-171), MBC-5or IMR-90 (ATCC, CCL-186).

Also, the term “normal cell” means a cell under a state withoutcancerating, namely a cell having a finite life span.

Whether a cell is a young cell or a senescent cell can be expressed, forexample, by a unit “PD”.

The term “PD” as used herein is the abbreviation of population doublingsand used as a unit which shows the number of cell divisions so farcarried out by the cell. In this connection, since the number of celldivisions of each cell is generally different, PD is an average value inan individual cell in a cell group. For example, in the case of humanfibroblast, the young cell generally means a cell of 0 to 50 PD(preferably a cell of 30 to 45 PD), and the senescent cell means a cellof 60 to 80 PD (preferably a cell of 60 to 65 PD).

As the host cell, both of the young cell and senescent cell can be used,but it is desirable to use a young cell capable of carrying out celldivision (that is, a cell which is not reaching the replication lifespan and keeping the ability to actively perform cell division). This isbecause the senescent phenotype which can be rapidly obtained by thegene introduction shown below is greatly different from the phenotypebefore the gene introduction and can therefore be easily distinguished.

The introduction gene which can be used in preparing the transformedcell of the present invention is not particularly limited, so long as itis a gene which can encode

(a) a protein capable of phosphorylating p38 protein (to be referred toas “p38 protein kinase” hereinafter), or

(b) p38 protein, a mutant of p38 protein, a kinase domain of p38protein, a kinase domain of p38 protein mutant or a fusion proteincontaining them (to be referred to as “p38 protein or a derivativethereof” hereinafter).

The p38 protein kinase (a) is not particularly limited with the provisothat it can phosphorylate p38 protein, but for example,

(a1) MKK6 protein or MKK3 protein which is known as a MAPK that directlyphosphorylates p38 protein in the p38 MAPK pathway;

(a2) an MKK6 protein mutant or an MKK3 protein mutant which is a naturalor artificial mutant of the MKK6 protein or MKK3 protein (a1) and inwhich the activity to phosphorylate p38 protein is maintained orenhanced;

(a3) respective kinase domain of the MKK6 protein or MKK3 protein (a1)or respective kinase domain of the MKK6 protein mutant or MKK3 proteinmutant (a2);

(a4) respective fusion protein of the MKK6 protein or MKK3 protein (a1)with a receptor ligand binding domain or respective fusion protein ofthe MKK6 protein mutant or MKK3 protein mutant (a2) with a receptorligand binding domain; or

(a5) respective fusion protein of respective kinase domain of the MKK6protein or MKK3 protein with a receptor ligand binding domain orrespective fusion protein of respective kinase domain of the MKK6protein mutant or MKK3 protein mutant with a receptor ligand bindingdomain can be cited.

In this description, the term “phosphorylates p38 protein” as usedherein means that it directly phosphorylates the p38 protein itself.

Examples of the aforementioned MKK6 protein or MKK3 protein (a1) includeMKK6 protein or MKK3 protein derived from a mammal (e.g., human, monkey,mouse, rat or hamster), and it is desirable to use human or mouse MKK6protein or MKK3 protein, it is more desirable to use human or mouse MKK6protein and it is particularly desirable to use human MKK6 protein. Forexample, the region consisting of the amino acid residues of from the53rd to 314th positions is the kinase domain in the case of the humanMKK6 protein, and the region consisting of the amino acid residues offrom the 35th to 269th positions is the kinase domain in the case of thehuman MKK3 protein.

Examples of the aforementioned MKK6 protein mutant or MKK3 proteinmutant (a2) include a natural allele mutant of the MKK6 protein or MKK3protein, a mutant prepared by artificially introducing a mutationthrough a known genetic engineering technique (e.g., site-specificmutagenesis; Proc. Natl. Acad. Sci. USA, 81: 5662-5666, 1984) and thelike. More illustratively, a constitutive active type mutant in whichthe serine at the 207th position (Ser 207) and threonine at the 211thposition (Thr 211) in the human MKK6 protein are respectivelysubstituted with glutamic acid (Glu; E) (Mol. Cell. Biol., 16:1247-1255, 1996) can be exemplified.

Examples of the respective kinase domain (a3) of MKK6 protein, MKK3protein or a mutant thereof include a human MKK6 protein fragmentcomprising a sequence consisting of the amino acid residues of from the53rd to 314th positions in the human MKK6 protein (preferably a humanMKK6 protein fragment consisting of a sequence consisting of the aminoacid residues of from the 53rd to 314th positions in the human MKK6protein), and a human MKK3 protein fragment comprising a sequenceconsisting of the amino acid residues of from the 35th to 269thpositions in the human MKK3 protein (preferably a human MKK3 proteinfragment consisting of a sequence consisting the amino acid residues offrom the 35th to 269th positions in the human MKK3 protein).

The receptor in the respective fusion protein (a4) of the MKK6 proteinor MKK3 protein or a mutant thereof with a receptor ligand bindingdomain, or respective fusion protein (a4) of respective kinase domain ofthe MKK6 protein or MKK3 protein or a mutant thereof with a receptorligand binding domain is not particularly limited, so long as it is areceptor which, when the aforementioned fusion protein is expressed in ahost cell, can activate another constituting component of the fusionprotein (e.g., the MKK6 protein or MKK3 protein or a mutant thereof, orrespective kinase domain of the MKK6 protein or MKK3 protein or a mutantthereof) through the activation of the receptor (that is, binding of aligand to the receptor ligand binding domain). Examples of the receptorinclude a steroid receptor (e.g., an estrogen receptor), and forexample, the ligand binding domain of a human estrogen receptor is aregion consisting of the amino acid residues of from the 282nd to 595thpositions of the human estrogen receptor.

Examples of the p38 protein or a derivative (b), include

(b1) p38 protein;

(b2) a p38 protein mutant which is a natural or artificial mutant of thep38 protein (b1) and in which phosphorylation ability of p38 protein(that is, activity of p38 protein to phosphorylate its downstreamprotein) is maintained or enforced;

(b3) kinase domain of the p38 protein (b1) or kinase domain of the p38protein mutant (b2);

(b4) respective fusion protein of the p38 protein (b1) or p38 proteinmutant (b2) with a receptor ligand binding domain; and

(b5) respective fusion protein of respective kinase domain (b3) of thep38 protein or p38 protein mutant with a receptor ligand binding domain.

Examples of the aforementioned p38 protein (b1) include p38 proteinderived from a mammal (e.g., human, monkey, mouse, rat or hamster), andit is desirable to use human p38 protein. For example, the regionconsisting of the amino acid residues of from the 24th to 388thpositions is the kinase domain in the case of the human p38 protein.

Examples of the aforementioned p38 protein mutant (b2) include a naturalallele mutant of the p38 protein, a mutant prepared by artificiallyintroducing a mutation through a known genetic engineering technique(e.g., site-specific mutagenesis) and the like.

Examples of the respective kinase domain (b3) of p38 protein or a p38protein mutant include a human p38 protein fragment consisting of asequence comprising the amino acid residues of from the 24th to 388thpositions in the human p38 protein (preferably a human p38 proteinfragment consisting a sequence consisting of the amino acid residues offrom the 24th to 388th positions in the human p38 protein).

In the respective fusion protein (b4) of the p38 protein or a p38protein mutant with a receptor ligand binding domain, or respectivefusion protein (b5) of respective kinase domain of the p38 protein or ap38 protein mutant with a receptor ligand binding domain, theaforementioned receptor is not particularly limited, so long as it is areceptor which, when the aforementioned fusion protein is expressed in ahost cell, can activate the p38 protein or a p38 protein mutant orrespective kinase domain thereof, as another constituting component ofthe fusion protein, through the activation of the receptor (that is,binding of a ligand to the receptor ligand binding domain) Examples ofthe receptor include a steroid receptor (e.g., an estrogen receptor).

The method for introducing an introduction gene into a host cell inpreparing the transformed cell of the present invention is notparticularly limited and can be carried out using a conventionally knowngene transduction method. Examples of the conventionally known genetransfer method include a method which uses a retrovirus (Proc. Natl.Acad. Sci. USA, 92: 9146-1950, 1995), a method in which an adenovirus isused, a DEAE-dextran method (Nucleic Acids Res., 11: 1295-1308, 1983), acalcium phosphate-DNA coprecipitation method (Virology,52:456-457,1973), a method which uses a commercially availabletransfection reagent (e.g., FuGENETM6 Transfection Reagent; manufacturedby Roche Diagnostics), an electroporation (EMBO J., 1: 841-845, 1982)and the like.

Since a gene coding for

(a) a protein capable of phosphorylating p38 protein, the kinase domainthereof or a fusion protein containing them, or

(b) p38 protein, the kinase domain thereof or a fusion proteincontaining them is introduced into the transformed cell of the presentinvention, cellular senescence is rapidly induced and its ability tocarry out cell division is lost, whereas it kept the ability to carryout cell division actively before the aforementioned gene introduction.

In this connection, when a gene coding for a fusion protein with areceptor is used as the introduction gene, it is necessary for theinduction of cellular senescence to allow the aforementioned receptorligand to react upon the transformed cell.

Since the transformed cell of the present invention can be preparedwithin a markedly short period of time in comparison with a naturalsenescent cell obtained via scores of times or more of natural division,it is very useful in constructing a screening system for anti-agingagents.

The screening method for the anti-aging agent of the present inventionincludes

(1) a step for allowing a normal senescent cell or a normal cellintroduced with a gene capable of inducing cellular senescence tocontact with a substance to be tested, and

(2) a step for analyzing an aging index in the cell.

Although the cell which can be used in the screening method of thepresent invention is not particularly limited, so long as it is a cellcapable of expressing p38, examples include a normal senescent cell, theaforementioned transformed cell and a transformed cell prepared bytransforming a normal cell with a gene coding for

(c) a protein positioned at the upstream of a MAPKK protein of the p38pathway, a mutant of the protein positioned at the upstream of the MAPKKprotein of the p38 pathway, the kinase domain of the protein positionedat the upstream of the MAPKK protein of the p38 pathway, or a fusionprotein containing them, or

(d) a protein which activates the p38 MAPK pathway or a fusion proteinthereof.

The introduction gene which can be used for the preparation of the abovetransformed cells (c) and (d) is not particularly limited, so long as itis a gene which can encode

-   (c) a protein positioned at the upstream of a MAPKK protein of the    p38 pathway, a mutant of the protein positioned at the upstream of    the MAPKK protein of the p38 pathway, the kinase domain of the    protein positioned at the upstream of the MAPKK protein of the p38    pathway, or a fusion protein containing them, or-   (d) a protein which can activate the p38 MAPK pathway.

The protein positioned at the upstream of a MAPKK protein of the p38pathway is not particularly limited, so long as it is a protein includedin the p38 MAPK pathway, which can directly or indirectly phosphorylatethe MAPKK protein of the p38 pathway, and its examples include proteinssuch as MAP3K, for example, MLK, MELK, ASK1, TAK1, and MTK1.

In addition, the MAPKK protein of the p38 MAPK pathway means a proteinwhich phosphorylates p38 proteins such as MMK3, MMK6 and MMK4.

According to the present invention, the protein which activates the p38MAPK pathway is not particularly limited, so long as it is a proteinwhich does not belong to the p38 MAPK pathway but activates the p38 MAPKpathway, and its examples include a cytokine which activates the p38MAPK pathway, MAPKKK kinases such as PAK1 and GCK, a protein containedin the classical MAPK pathway, mutants thereof and fusion proteinsthereof, of which Raf and a fusion protein containing Raf can be citedas preferred examples.

Although they are not particularly limited, examples of the substance tobe tested which can be used in the screening method of the presentinvention include commercially available compounds, variousconventionally known compounds, a group of compounds obtained by thecombinatorial chemistry techniques (Tetrahedron, 51: 8135-8173, 1995),culture supernatants of microorganisms, natural components derived fromplants and marine organisms, animal tissue extracts, or compoundsobtained by chemically or biologically modifying a compound selected bythe screening method of the present invention.

According to the screening method of the present invention, the methodfor allowing a normal senescent cell or a normal cell introduced with agene capable of inducing cellular senescence to contact with a substanceto be tested is not particularly limited. However, for example, it canbe carried out by adding the substance to be tested to a cultured mediumof the aforementioned cell, by replacing a general medium (namely amedium which does not contain the substance to be tested) with a mediumto which the substance to be tested was added in advance, or by addingthe aforementioned cell to a medium to which the substance to be testedwas added in advance. In this connection, when a gene coding for afusion protein with a receptor is used as the transferring gene, it isnecessary to induce cellular senescence prior to contacting with asubstance to be tested, by allowing the aforementioned receptor ligandto act upon the transformed cell.

According to the screening method of the present invention, whether ornot the aforementioned substance to be tested has an anti-aging activitycan be judged by allowing a normal senescent cell or a normal cellintroduced with a gene capable of inducing cellular senescence tocontact with a substance to be tested and then observing change in anaging index in the aforementioned cell. In that case, it is desirable tojointly use, as a control, a normal senescent cell or a normal cellintroduced with a gene capable of inducing cellular senescence, which isnot contacted with the substance to be tested, because change in theaging index becomes more clear.

The term “anti-aging activity” as used herein means an anti-cellularaging activity, and the term “cellular senescence” means a cellularsenescence due to the span of life of division, namely arrest of cellgrowth due to the span of life of cell division.

The aforementioned “anti-cellular aging activity” includes, for example,

(1) a cellular replicative regeneration activity which regenerates theability to perform cell division in a senescent cell which lost the celldivision ability, and

(2) a cellular senescence delaying activity which delays lowering rateof the cell division ability.

As the “aging index” which can be observed by the screening method ofthe present invention, it is not particularly limited so long as it isan index which shows the presence or absence, or the degree of cellularsenescence, and examples include cell growth rate, a morphologicalchange of cytoplasm, a change of an enzyme activity specific forcellular senescence, ratio of S phase cell and the like.

Specifically, in case that the cell growth rate is analyzed as the agingindex, it can be judged that the aforementioned substance to be testedhas the anti-aging activity when the cell growth rate after contactingwith the substance to be tested becomes quick in comparison with acontrol (a aging cell which is not contacted with the substance to betested).

In the case where a morphological change of cytoplasm is analyzed as theaging index, since the cytoplasm of a senescent cell generally takes aflat morphology, it can be judged that the aforementioned substance tobe tested has the anti-aging activity when the flat cytoplasm becomesthin after its contact with the substance to be tested.

When a change of cellular senescent specific enzyme activity is analyzedas the aging index, β-galactosidase can be cited as the cellularsenescence-specific enzyme.

In the case where the activity of a cellular senescence-specific enzyme(e.g., β-galactosidase) is reduced when contacted with a substance to betested in comparison with a control (a senescent cell un-contacted withthe substance to be tested), it can be judged that the aforementionedsubstance to be tested has the anti-aging activity.

In the case where the ratio of the S phase cell is analyzed as the agingindex, since the ratio of S phase cell is generally high in the group ofcells having active cell growth, when the ratio of S phase cell aftercontacting with the substance to be tested becomes high in comparisonwith a control (a senescent cell un-contacted with the substance to betested), it can be judged that the aforementioned substance to be testedhas the anti-aging activity.

Since a substance obtained by the screening method of the presentinvention has the anti-aging activity, namely a cellular replicativeregeneration activity and/or a cellular senescence delaying activity, itis useful as the active ingredient of a cellular replicativeregenerating agent or a cellular senescence delaying agent.

More specifically, the substance obtained by the screening method of thepresent invention has, for example, a p38 protein inhibitory activity oran activity to inhibit a cellular senescence signal transductionmolecule which constitutes the cellular senescence signal transductioncascade at a down stream of the p38 protein.

The anti-aging agent of the present invention contains a p38 proteininhibitor as the active ingredient.

Various compounds are known as the p38 protein inhibitor, and itsexamples include the pyrazole compounds described in WO 95/31451, thepyrimidine compounds described in WO 97/33883 and thenitrogen-containing heterocyclic derivatives described in WO 98/27098.

As the conventionally known p38 protein inhibitors, examples includeVX-745, VX-954, VX-702, SB203580“4-[4-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-5-yl]-pyridine”,SB220025, RWJ-68354, RPR-68354, RPR-200765A, SC-XX906, HEP-689(SB235699) and LL-Z-16402.

With regard to the p38 protein inhibitor as the active ingredient of theanti-aging agent of the present invention, preferred is a compoundrepresented by formula (1)

[wherein R₁ is a pyridyl group which is optionally substituted with oneor two substituents independently selected from the group consisting ofan alkyl group having from 1 to 4 carbon atoms, a halogen atom, analkoxy group having from 1 to 4 carbon atoms, an alkylthio group havingfrom 1 to 4 carbon atoms, an amino group and a mono- or di-alkyl (thenumber of carbon atoms of each alkyl moiety=1 to 6)amino group; R₂ is ahydrogen atom or an alkyl group having from 1 to 10 carbon atoms; R₃ isa phenyl group which is optionally substituted with one to threesubstituents independently selected from the group consisting of ahalogen atom, an alkyl group having from 1 to 5 carbon atoms and—(CH₂)_(n)Y; R₄ is a phenyl group which is optionally substituted withone or two halogen atoms; Y is —S(O)_(m)R₅ or —NR₆R₇; R₆ is a hydrogenatom or an alkyl group having from 1 to 10 carbon atoms, R₇ is ahydrogen atom, a benzyl group or an alkyl group having from 1 to 10carbon atoms, or R₆ and R₇ together with the nitrogen atom to which theyare bound may form a six-membered heterocyclic ring which optionally hasan oxygen atom; R₅ is an alkyl group having from 1 to 10 carbon atoms; mis 0, 1 or 2; and n is 0 or 1].

In addition, with regard to the p38 protein inhibitor as the activeingredient of the anti-aging agent of the present invention, a compoundof the aforementioned formula (1) wherein R₁ is an unsubstituted4-pyridyl group, R₂ is a hydrogen atom, R₃ is a phenyl group substitutedwith —S(O)_(m)R₅ (wherein m is 1 or 2, and R₅ is an alkyl group havingfrom 1 to 4 carbon atoms) and R₄ is a phenyl group substituted with ahalogen atom is preferable; a compound wherein the substituent groups inR₃ and R₄ are bound at respective para-positions is more preferable; acompound wherein R₃ is a phenyl group substituted with —SOR₅ (wherein R₅is an alkyl group having from 1 to 4 carbon atoms) and R₄ is a phenylgroup substituted with a fluorine atom, a chlorine atom or an iodineatom is still more preferable; and a compound wherein R₃ is a phenylgroup substituted with —SOCH₃ and R₄ is a phenyl group substituted withfluorine atom (namely SB203580) is particularly preferable.

A cellular replicative regenerating agent and a cellular senescencedelaying agent are included in the anti-aging agent of the invention.

Examples of the cellular replicative regenerating agent include a woundtreating agent which accelerates restoration of a wounded part; apreventing and/or treating agent for arteriosclerosis (particularlyarteriosclerosis obliterans) wherein fallout and/or damage of vascularendothelial cells and the like could become the cause; a growth enhancerfor tissue culture in regenerative treatment; a liver functionrecovering agent for hepatic cirrhosis and the like; an anemia improvingagent for senile anemia and the like; an immunity recovering agent forsenile immunodeficiency and the like; a mucosal epithelium functionrecovering agent for a chronic inflammatory gastrointestinal disease andthe like; and a trichogenous agent, a hair tonic and the like.

Examples of the cellular senescence delaying agent include anarteriosclerosis preventing agent; a liver function reduction preventingagent for hepatic cirrhosis and the like; an anemia preventing agent forthe aged and the like; an immunity reduction preventing agent for theaged and the like; a mucosal epithelium function reduction preventingagent for a chronic inflammatory gastrointestinal disease and the like;and an alopecia preventing agent, a hair tonic and the like.

The anti-aging agent of the invention can be prepared as apharmaceutical composition in accordance with a general method using anappropriate pharmaceutical carrier. As the carrier, various carrierswidely used in general drugs, such as an excipient, a binder, adisintegrator, a lubricant, a coloring agent, a taste masking agent, aflavoring agent, a surfactant and the like, can be used.

Dosage form of the anti-aging agent of the present invention is notparticularly limited and can be optionally selected in response to thetreating purpose. Examples include parenteral preparations such asinjections, suppositories and external preparations (ointments, adhesivepreparations and the like); aerosols and the like; and oral preparationssuch as tablets, powders, fine granules, granules, capsules, solutions,pills, suspensions, syrups and emulsions.

The aforementioned various drugs can be made into pharmaceuticalpreparations by general methods.

Examples of carriers which can be used in forming oral solidpreparations such as tablets, powders, fine granules and granulesinclude an excipient (lactose, sucrose, sodium chloride, glucose,starch, calcium carbonate, kaolin, crystalline cellulose, anhydrousdibasic calcium phosphate, alginic acid or the like); a binder (simplesyrup, glucose solution, starch solution, gelatin solution; polyvinylalcohol, polyvinyl ether, polyvinyl pyrrolidone, carboxymethylcellulose,shellac, methyl cellulose, ethyl cellulose, sodium alginate, gum Arabic,hydroxypropylmethylcellulose, hydroxypropylcellulose, water and/orethanol solution thereof or the like); a disintegrator (starch, alginicacid, crosslinked polyvinyl pyrrolidone, crosslinkedcarboxymethylcellulose sodium, carboxymethylcellulose calcium, starchsodium glycolate or the like); a release controlling agent (a higherfatty acid, a higher aliphatic alcohol, cacao butter, a hydrogenatedoil, a water-soluble polymer, a gastric polymer, an enteric polymer orthe like); an absorption enhancing agent (a surfactant such as aquaternary ammonium salt, sodium lauryl sulfate or sorbitan monooleate);an adsorbing agent (starch, lactose, kaolin, bentonite, silicic acidanhydride, hydrous silicon dioxide, magnesium aluminometasilicate,colloidal silicic acid or the like); and a lubricant (purified talc, astearate, a silicic acid, polyethylene glycol or the like).

If necessary, tablets can be made into general coating-treated tabletssuch as sugar coated tablets, gelatin coated tablets, gastric coatedtablets, enteric coated tablets or water-soluble film coated tablets.

Capsules can be prepared by mixing with various carriers exemplified inthe above and filling in hard gelatin capsules or soft capsules.

Liquid preparations may be water-soluble or oil-soluble suspensions,solutions, syrups or elixirs, and these can be prepared in accordancewith a conventional known method using general additive agents.

When formed into a shape of suppositories, polyethylene glycol, cacaobutter, lanolin, a higher alcohol, an ester of a higher alcohol,gelatin, hard fat or the like can be used as the carrier by adding anappropriate absorption enhancing agent thereto.

When formed into a form of injections, examples of useful carriersinclude a diluent (water, ethyl alcohol, Macrogol, propylene glycol orthe like); a pH adjusting agent or buffer agent (citric acid, aceticacid, phosphoric acid, lactic acid or a salt thereof; sulfuric acid,sodium hydroxide or the like); and a stabilizing agent (sodiumpyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid,thiolactic acid or the like). In addition, in this case, sodiumchloride, glucose, mannitol or glycerol can be contained in thepharmaceutical preparation, in an amount sufficient for preparing anisotonic solution, and a general solubilization assisting agent, asoothing agent, a local anesthetic or the like can also be added.

When prepared into a form of ointments such as pastes, creams or gels,generally used bases, stabilizing agents, moistening agents,preservatives or the like are blended as occasion demands, and they canbe mixed and made into a pharmaceutical preparation by a conventionalmethod. Examples of the useful bases include white petrolatum,polyethylene, paraffin, glycerin, a cellulose derivative, polyethyleneglycol, a dimethyl polysiloxane, a carboxyvinyl polymer, bentonite andthe like. As the preservatives, parahydroxybenzoate ester and the likecan for example be used.

When adhesive preparations are produced, the aforementioned ointments,creams, gels, pastes or the like can be coated on a general support by aconventional method. As the support, cotton; staple; woven or nonwovenfabric made of chemical fibers; and film such as soft vinyl chloride,polyethylene and polyurethane.

Although administration method of the anti-aging agent of the presentinvention is not particularly limited, it can be optionally administeredorally or parenterally in response, for example, to the dosage form ofthe preparation, age, sex or other conditions of each patient or thedegree of symptoms of the patient.

Clinical dose of the active ingredient of the anti-aging agent of thepresent invention can be optionally selected in response, for example,to the administration method, age and sex of each patient, form of thedisease and other conditions, but it can be administered generally at adose of from 1 to 2,000 mg per day per adult or dividing the daily doseinto several doses.

EXAMPLES

The present invention is explained in detail based on reference examplesand examples; however, they do not limit the scope of the presentinvention.

Reference Example 1

(1) Culturing of Cell

In this reference example, using a human normal fibroblast WI-38(purchased from JCRB cell bank), confirmation was made on thedifferences in various aging indexes between active proliferating youngcell which carried out 42 times of cell division after collection of thecell and its senescent cell after 61 times of cell division.

In this case, the aforementioned cells were cultured using 10% fetalcalf serum (FCS)-containing DMEM (Dulbecco's modified Eagle's medium),and their sub-culturing was carried out at a ratio of 1:4. In that case,one sub-culturing was defined as 2 PD. According to this, a cell of from30 to 40 PD was used as the young cell, and a cell of 60 PD as thesenescent cell.

(2) Detection of Cellular Senescence-specific β-galactosidase(Senescence-associated β-galactosidase) Activity

The cellular senescence-specific β-galactosidase activity was detectedby the method of Dimri et al. (Proc. Natl. Acad. Sci. USA, 92:9363-9363, 1995).

Specifically, young and senescent cells of the human normal fibroblastWI-38 were respectively washed with PBS and then fixed using 0.5%glutaraldehyde aqueous solution. After the fixation, the cells weresoaked in a staining solution [prepared by dissolving 1 mg/ml of X-gal(5-bromo-4-chloro-3-indolyl-β-galactosidase), 5 mmol//L potassiumferrocyanide, 5 mmol//L potassium feriocyanide and 1 mmol//L magnesiumchloride in phosphate buffered saline (PBS; pH 6.0)] and allowed toreact at 37° C. for 5 hours or more.

Photomicrographs of the respective stained cells are shown in FIG. 1. InFIG. 1, the photograph (a) is a result of a young cell (42 times ofdivision), and the photograph (b) is a result of a senescent cell (61times of division). Each cell is stained blue in reality when it has theβ-galactosidase activity, but it is shown in black in the monochromephotograph shown in FIG. 1.

As shown in FIG. 1, in the senescent cell [photograph (b)], thecytoplasm was flat in comparison with the young cell [photograph (a)],and the β-galactosidase activity was confirmed.

(3) Confirmation of Phosphorylated State of p38 Protein

In this reference example, the phosphorylated state of p38 protein inyoung and senescent cells of the human normal fibroblast WI-38 wasconfirmed by a Western blotting respectively using an antibody capableof detecting total p38 protein and another antibody capable ofspecifically detecting a phosphorylation type p38 protein (namely activetype p38 protein) alone.

Specifically, young and senescent cells of the human normal fibroblastWI-38 (about 5×10⁶ cells) cultured in the same manner as in ReferenceExample 1(1) were respectively dissolved in 0.1 ml of a buffer solutionfor dissolution [lysis buffer; 150 mmol//L sodium chloride, 0.5% NP-40,50 mmol//L Tris-HCl (pH 7.5), 3 mmol//L EDTA, 3 mmol//L EGTA, 3 mmol//Lglycerophosphate, 0.1μ//L Na₃VO₄, 1 μg/ml leupeptin and 1 mmol//Lphenylmethanesulfonyl fluoride (PMSF)].

Subsequently, each extraction sample corresponding to 30 μg as theamount of protein was separated by an SDS polyacrylamide gelelectrophoresis in the usual way and then transferred on a membrane(Immobilon-P membrane; manufactured by Millipore). After subjecting themembrane to a blocking treatment using a commercially available blockingagent (Block Ace; manufactured. by Dainippon Pharmaceutical), a primaryantibody reaction and a secondary antibody reaction were carried out inorder.

In this case, a rabbit anti-p38 antibody (manufactured by NEB; anantibody which reacts with all p38 proteins regardless of the presenceor absence of phosphorylation) and a rabbit anti-phosphorylated p38antibody (manufactured by NEB; an antibody which reacts withphosphorylated p38 protein but does not react with un-phosphorylated p38protein) were used as the primary antibodies, and a horseradishperoxidase-labeled anti-rabbit IgG antibody (manufactured by Amersham)was used as the secondary antibody. Detection of the peroxidase activitywas carried out using commercially available detection kit (manufacturedby ECL; Amersham) and X-ray film (manufactured by Amersham).

The results are shown in FIG. 2. In this connection, a result of ananisomycin-treated young cell (42 PD) is also shown as a positivecontrol. It is known that p38 protein is activated (phosphorylated) whena cell is treated with anisomycin. Anisomycin treatment of the cell wascarried out prior to its lysis with the lysis buffer by culturing it for30 minutes in DMEM containing 30 μmol//L anisomycin and 10% FCS.

In FIG. 2, lane 1 shows a result of the young cell (42 PD), lane 2 showsa result of the senescent cell (61 PD), and lane 3 shows a result of thepositive control (namely, the young cell treated with anisomycin).

As shown in FIG. 2, difference in the expression of total p38 proteinwas not found among the three types of cells. On the other hand, theactive type p38 protein was less in the young cell, increased in theanisomycin-treated young cell and more significantly increased in thesenescent cell. Accordingly, it was shown that the p38 protein isactivated by the cellular senescence of human normal fibroblast causedby its replicative life span.

Example 1

(1) Effect of p38 Protein Inhibitor in Human Normal Fibroblast

In the example, effect of a p38 protein inhibitor SB203580 was comparedusing the same young cell (42 times of division) and senescent cell (61times of division) of the human normal fibroblast WI-38 used inReference Example 1. Specifically, the same procedure of the followingExample 2 (2) was repeated, except that a young cell (42 times ofdivision) and a senescent cell (61 times of division) of the humannormal fibroblast WI-38 cultured in the same manner in Reference Example1(1) were used instead of the transformed cell.

The results are shown in FIG. 3. In FIG. 3, the polygonal line a is cellgrowth curve of the young cell in the presence of SB203580, thepolygonal line b is cell growth curve of the young cell in the absenceof SB203580, the polygonal line c is cell growth curve of the senescentcell in the presence of SB203580 and the polygonal line d is cell growthcurve of the senescent cell in the absence of SB203580. The symbol “+SB”in FIG. 3 means that it was treated with 10 μmol//L of SB203580.

Also, the ratio of S phase cells in each treated group was determined byFACS analysis in accordance with the procedure described in thefollowing Example 2(3-a), and the results shown in FIG. 4. The symbol“+” in FIG. 4 means that it was treated with 10 μmol//L of SB203580.

In addition, cell morphology and β-galactosidase activity in eachtreated group were analyzed in accordance with the procedure describedin Reference Example 1(2).

The young cell grew well (doubling time=about 2 days) regardless of thepresence or absence of SB203580 as shown in FIG. 3 (polygonal line a andpolygonal line b), and its S phase cell was about 20% as shown in FIG.4. The SB203580-untreated senescent cell grew slowly (doublingtime=about 6 days) (cf. polygonal line d in FIG. 3), its S phase cellwas about 5% (cf. FIG. 4). Although illustrative data are not shown, itshowed a morphologically characteristic of a cellularsenescence-specific β-galactosidase-positive flat cytoplasm. On theother hand, the SB203580-treated senescent cell showed acceleratedgrowth in comparison with the untreated case (cf. polygonal line c inFIG. 3), the S phase cell shown by 72 hours of bromodeoxyuridine (BrdU)labeling was significantly increased to about 25% (cf. FIG. 4), and itshowed a morphologically characteristic in that the cellularsenescence-specific β-galactosidase-positive degree was low and the flatcytoplasm became thin, thus resembling the young cell. Based on theseresults, it was shown that the SB203580 as a compound having p38 proteininhibitory activity partially restarts cell growth of a cell reachingits replicative life span.

Example 2

(1) Preparation of Transformed Cell of the Present Invention

In the example, the transformed cell of the present invention wasprepared in accordance with the procedure described as follows, byintroducing a human MKK6 gene coding for the human MKK6 protein, or anMKK6EE gene coding for a constitutive active type mutant of the MKK6 (tobe referred to as MKK6EE protein hereinafter), into a young cell of thehuman normal fibroblast WI-38 by a gene introduction method which uses aretrovirus.

In this connection, the MKK6 protein is an MAPK kinase (MAPKK) whichphosphorylates and activates p38 protein. Also, the MKK6EE protein is amutant of the MKK6 protein in which serine at the 207th position (Ser207) and threonine at the 211th (Thr 211) are respectively substitutedwith glutamic acid (Glu; E) (Mol. Cell. Biol., 16: 1247-1255, 1996).

The MKK6 gene was obtained by amplifying a DNA fragment of about 1 kbpby a PCR method which uses a plasmid pSRα-MKK6 (J. Biol. Chem., 271:13675-13679, 1996) as the template and a combination of a forward primerconsisting of the nucleotide sequence represented by SEQ ID NO:1 and areverse primer consisting of the nucleotide sequence represented by SEQID NO:2. In this case, the aforementioned PCR was carried out byincubating at 94° C. (5 minutes), repeating a cycle of 94° C. (30seconds), 52° C. (30 seconds) and 72° C. (1 minute) 15 times and thenfinally incubating at 72° C. (10 minutes). Also, the MKK6EE gene wasobtained by modifying the aforementioned MKK6 gene using a geneticengineering technique in accordance with the conventional method, namelysite-specific mutagenesis (Proc. Natl. Acad. Sci. USA, 81: 5662-5666,1984).

Each of the thus obtained MKK6 gene and MKK6EE gene was subcloned into apuromycin-resistant gene-containing retrovirus vector pMXpuro (Exp.Hematol., 24: 324-329, 1996).

The gene introduction by retrovirus was carried out in accordance with aconventionally known method (Proc. Natl. Acad. Sci. USA, 92: 9146-9150,1995). That is, a packaging cell NX cell [4×10⁶ cells/10 cm dish (J.Virol., 38: 895-905, 1981)] was transfected with 6 μg of a retrovirusDNA containing each gene (or the retrovirus vector pMXpuro as acontrol). After 48 hours, the culture supernatant was recovered and usedas a virus solution.

As the target cell, young cell (42 PD; 5×10⁵ cells/10 cm dish) of thehuman normal fibroblast WI-38 was cultured for 8 hours in the virussupernatant to which polybrene (final concentration=8 μg/ml) had beenadded. The virus supernatant was once removed, and then fresh virussupernatant [containing polybrene (final concentration=8 μg/ml)] wasagain added, followed by the culturing for 8 hours. Each of theinfection-treated cells was cultured for 3 days in DMEM containingpuromycin (2.5 μg/ml).

On the 4th day after carrying out the infection, the medium was changedto DMEM containing 2.5% dimethyl sulfoxide (DMSO), followed by theculturing for 6 days. In this case, the medium exchange was carried outevery day during the culturing for 6 days. The relative number of cellsduring these 6 days was measured every other day using a commerciallyavailable kit [Cell Counting Kit (WST-1 assay), manufactured. byDOJINDO].

The WST-1 assay is a method to know indirectly degree of cell growthability with the use of the calorimetric determination of the activityof dehydration reactions wherein intracellular lactate dehydrogenase andthe like are related.

Measurement and calculation of the relative cell number werespecifically carried out by the following method. That is, 1,000 cellsper each well of a 96 well plate were precultured for 24 hours. Afteradding the WST-1 reagent to the culture medium, the coloring reactionwas carried out for 4 hours in a CO₂ incubator. After the reaction,measurement was carried out using a plate reader (measuring wavelength=450 nm). These were measured on three samples on each of themeasuring days (on the 0th, 2nd, 4th and 6th days) during the growthperiod, and the average values were used as the data of respectivemeasuring days. The relative number of cells of measured values onrespective measuring days was calculated by defining the average ofmeasured values on the 0th day as 1. The results are shown in FIG. 5(a).

In FIG. 5, the 4th day after carrying out the infection treatment wasused as the “0th day”. Also, in FIG. 5, the symbol “WT” indicates thatit is a result of the MKK6 gene-introduced cell of the presentinvention, the symbol “EE” indicates that it is a result of the MKK6EEgene-introduced cell of the present invention, and the symbol “V”indicates that it is a result of the vector (retrovirus vector pMXpuro)alone-introduced cell (control).

As shown in FIG. 5( a), cell growth rates of the MKK6-expressing cell(WT) of the present invention and the MKK6EE-expressing cell (EE) of thepresent invention were significantly reduced on the 4th day, incomparison with the control cell (V) infected with the vector alone. Inaddition, in the case of the cell (EE) of the present inventionexpressing the MKK6EE gene, its growth was completely stopped before thecell became confluent.

(2) Effect of p38 Protein Inhibitor on the Transformed Cell of thePresent Invention

In the example, effect of the p38 protein inhibitor SB203580 on the twospecies of the transformed cell of the present invention obtained inExample 2(1) was confirmed.

Specifically, the retrovirus infection treatment described in Example2(1) and culturing of the cells obtained by the treatmentdescribed-above were repeated, except that the medium was replaced byDMEM containing 10 μmol//L SB203580 (manufactured. by Sigma) on the 4thday after carrying out the infection treatment. The results are shown inFIG. 5( b).

The symbol “+SB” in FIG. 5( b) indicates that it is a result of the caseof adding SB203580 to the medium. In this connection, the symbol “−SB”in FIG. 5( a) indicates that it is a result of the case where SB203580was not added to the medium.

With regard to the growth inhibitory effect by the MKK6 gene or MKK6EEgene shown in FIG. 5( a), it was inhibited by the p38 protein inhibitorSB203580 as shown in FIG. 5( b).

In addition, although illustrative data were not shown, the SB203580also showed the anti-aging activity upon a senescent cell (61 PD) of thehuman normal fibroblast WI-38 cultured in the same manner as inReference Example 1(1).

(3) Confirmation of Various Aging Indexes in the Transformed Cell of thePresent Invention

(3-a) Ratio of S Phase Cell by FACS Analysis

Using the two species of the transformed cell of the present inventionand one species of a control cell wherein the vector alone wasintroduced, obtained by repeating the same procedure of Example 2(1),the ratio of S phase cell was confirmed by a fluorescence-activated cellsorter (FACS) analysis. As the cells, cells after a culturing period of6 days, namely the cells of “during 6 days” in FIG. 5, were used.

Specifically, each of the cells after a culturing period of 6 days wassubjected to labeling in DMEM medium containing 10μmol//Lbromodeoxyuridine (BrdU) for 4 hours. Next, the cells were fixedwith 70% ethanol and treated with 2 mol//L hydrochloric acid for 30minutes. Subsequently, they were neutralized with 0.1 mol//L sodiumborate and then allowed to react with FITC-labeled anti-BrdU antibody(manufactured by BECTON DICKINSON) for 1 hour. After completion of thereaction, the cells were washed with a washing buffer [PBS containing0.5% Tween 20 and 1% bovine serum albumin (BSA)] and suspended in a PBSsolution containing 5 μg/ml ProPidium Iodide (PI). The samples wereanalyzed by a two dimensional flow cytometry. The results are shown inFIG. 6.

The symbol “Vector” in FIG. 6( a) indicates that it is a result of thevector (retrovirus vector pMXpuro) alone-introduced cell (control), thesymbol “MKK6WT” in FIG. 6( b) indicates that it is a result of the MKK6gene-introduced cell of the present invention, and the symbol “MKK6EE”in FIG. 6( c) indicates that it is a result of the MKK6EEgene-introduced cell of the present invention. Each ordinate in FIG. 6shows the degree of BrdU staining, and each abscissa shows the degree ofPI staining.

As shown in FIG. 6, the ratio of S phase cell in the MKK6-expressingcell (MKK6WT) of the present invention and the MKK6EE-expressing cell(MKK6EE) of the present invention was significantly reduced incomparison with the control cell (Vector) infected with the vectoralone.

(3-b) Cellular Senescence-specific β-galactosidase Activity

The procedure described in Reference Example 1(2) was repeated, exceptthat the two species of the transformed cell of the present inventionand one species of a control cell wherein the vector alone wasintroduced, obtained by repeating the same procedure of Example 2(1),(cells after a culturing period of 6 days in each case, namely the cellsof “on the 6th day” in FIG. 5) were used. The results are shown in FIG.7.

The symbol “Vector” in FIG. 7( a) indicates that it is a result of thevector (retrovirus vector pMXpuro) alone-introduced cell (control), thesymbol “MKK6WT” in FIG. 7( b) indicates that it is a result of the MKK6gene-introduced cell of the present invention, and the symbol “MKK6EE”in FIG. 7( c) indicates that it is a result of the MKK6EEgene-introduced cell of the present invention.

As shown in FIG. 7, the MKK6-expressing cell (MKK6WT) of the presentinvention and the MKK6EE-expressing cell (MKK6EE) of the presentinvention showed a flat morphology and, what is more, their cellularsenescence-specific β-galactosidase activity was considerably increased.

Next, the procedure described in Reference Example 1 (2) was repeated,except that the two species of the transformed cell of the presentinvention and one species of a control cell wherein the vector alone wasintroduced, obtained by repeating the same procedure of Example 2(1),(cells after a culturing period of 6 days in each case;SB203580-untreated cells) and the two species of the transformed cell ofthe present invention and one species of a control cell wherein thevector alone was introduced, obtained by repeating the same procedure ofExample 2(2), (cells after a culturing period of 6 days in each case;SB203580-treated cells) were used. Then 200 cells were optionallyselected under a microscope from each of independent three samples pereach group, and the ratio of β-galactosidase positive cells wascalculated by counting the number of β-galactosidase positive cellscontained therein. The results are shown in FIG. 8.

In FIG. 8, the symbol “V” indicates that it is a result of the vector(retro virus vector pMXpuro) alone-introduced cell (control), the symbol“WT” indicates that it is a result of the MKK6 gene-introduced cell ofthe present invention, and the symbol “EE” indicates that it is a resultof the MKK6EE gene-introduced cell of the present invention. Also, inthe “SB” column shown on the abscissa of FIG. 8, the symbol “+”indicates that it is a result of the SB203580-treated cells, and thesymbol “−” indicates that it is a result of the SB203580-untreatedcells.

As shown in FIG. 8, the increase of the cellular senescence-specificβ-galactosidase positive cells is inhibited by SB203580.

(3-c) Phosphorylated State of p38 Protein

The procedure described in Reference Example 1(3) was repeated, exceptthat the two species of the transformed cell of the present inventionand one species of a control cell wherein the vector alone wasintroduced, obtained by repeating the same procedure of Example 2(1),(cells after a culturing period of 6 days in each case) were used, andthat an anti-MKK6 antibody (sc-1992; manufactured by Santa Cruz), ananti-p16 antibody (sc-468; manufactured by Santa Cruz), an anti-p21antibody (sc-817; manufactured by Santa Cruz), an anti-p53 antibody(sc-263; manufactured by Santa Cruz) and an anti-actin antibody(manufactured by Boehringer-Mannheim) were used as the primaryantibodies. In this connection, the p16 protein and p21 protein areknown proteins as proteins characteristic of senescent cells. Also,actin is a protein which is known to have no difference in itsexpression quantity between young cells and senescent cells. The resultsare shown in FIG. 9.

In FIG. 9, the symbol “mock” indicates that it is a result of the vector(retrovirus vector pMXpuro) alone-introduced cell (control), the symbol“MKK6WT” indicates that it is a result of the MKK6 gene-introduced cellof the present invention, and the symbol “MKK6EE” indicates that it is aresult of the MKK6EE gene-introduced cell of the present invention.

As shown in FIG. 9, a large amount of the MKK6 protein was expressed inthe MKK6 or MKK6EE gene-introduced cell in comparison with the control.While expressed amount of the total p38 protein did not vary among thethree species of cells, expressed amount of the active type p38 proteinsignificantly increased in the MKK6 gene-introduced cell and markedlyincreased in the MKK6EE gene-introduced cell in comparison with thecontrol. In addition, expressed amount of each of the senescentcell-specific p16 protein and p21 protein increased in the MKK6 orMKK6EE gene-introduced cell. It was found based on these results thatcellular senescence is directly induced into young cells by theactivation of p38 protein.

Example 3

It was already reported that human normal fibroblast rapidly shows acellular senescence phenotype by the forced expression of activated Ras(Cell, 88: 1593-602, 1997). It was shown that this process is inducedvia the classical MAPK, namely Raf-Mek-Erk, which is in the downstreamof Ras (Genes Dev., 12: 3008-3019, 1998, Genes Dev., 12: 2997-3007,1998). By the use of this, Zhu et al. have reported a system forinducing cellular senescence by activating Raf with estrogen ortamoxifen (Genes Dev., 12: 2997-3007, 1998). This is a result of fusingan estrogen receptor gene with a sequence coding for the kinase regionof activated Raf gene.

(1) In accordance with the aforementioned method, induction of cellularsenescence was tested by carrying out gene transfer into the WI-38 celland thereby constructing a system which can induce cellular senescenceby the administration of tamoxifen.

The results are shown in FIG. 10 and FIG. 11. By the administration of 1nM or more of tamoxifen, termination of cell growth and increase ofcellular senescence-specific β-galactosidase positive cell wereobserved, there by confirming that the cellular senescence was induced.FIG. 10 shows the changes in the morphology of cells by variedadministration concentration of tamoxifen (4-HT), and FIG. 11 shows arelationship between concentration of administered tamoxifen (4-HT) andthe relative number of cells wherein the initial number of cells isdefined as 1.

(2) Using the system constructed in (1), activation of Raf was inducedby 10 nM of tamoxifen, and by simultaneously adding 25 μM of PD98059(Mek inhibitor) and SB203580 (p38 inhibitor), their effects on theRaf-induced cellular senescence induction was observed. The results areshown in FIG. 12, FIG. 13, FIG. 14 and FIG. 15.

FIG. 12 shows the ratio of cell growth in respective cells afterinduction of the activated Raf. In FIG. 12, tamoxifen is shown by asymbol “4-HT”, and the 25 μM PD98059-administered system is shown by asymbol “+PD”.

FIG. 13 shows the ratio of β-galactosidase positive cell in respectivecells after induction of the activated Raf. In FIG. 13, tamoxifen isshown by a symbol “4-HT”, and the 25 μM PD98059-administered system isshown by a symbol “+”.

FIG. 14 shows the ratio of cell growth in respective cells afterinduction of the activated Raf. In FIG. 14, tamoxifen is shown by asymbol “4-HT”, and the 25 μM SB203580-administered system is shown by asymbol “+SB”.

FIG. 15 shows the ratio of β-galactosidase positive cell in respectivecells after induction of the activated Raf. In FIG. 15, tamoxifen isshown by a symbol “4-HT”, and the 25 μM SB203580-administered system isshown by a symbol “+”.

PD98059 partially inhibited the termination of cell growth and increaseof cellular senescence-specific β-galactosidase positive cell induced bythe activation of Raf. However, SB203580 inhibited the termination ofcell growth and increase of cellular senescence-specific β-galactosidasepositive cell induced by the activation of Raf, more strongly than thePD98059. It was clarified by this information that both of the classicalMAPK and a stress-inducing MAPK p38 are concerned in the cellularsenescence induced by the activation of Raf, but the concern of p38 isparticularly large.

INDUSTRIAL APPLICABILITY

The transformed cell of the present invention is a cell useful forconstructing the screening system of the present invention, and,according to the screening method of the present invention, a substanceeffective as an anti-aging agent can be screened. In addition, acompound selected by the screening method of the present invention isuseful as an anti-aging agent.

The invention claimed is:
 1. An isolated senescent cell prepared by introducing and expressing an exogenous gene in a juvenile, non-transformed, mammalian cell; wherein said gene codes for a protein that directly phosphorylates p38 protein.
 2. The isolated senescent cell of claim 1, wherein said juvenile, non-transformed, mammalian cell is a human, monkey, mouse, rat or hamster cell.
 3. The isolated senescent cell of claim 2, wherein said juvenile, non-transformed, mammalian cell is a human cell.
 4. The isolated senescent cell of claim 1, wherein said juveni1e, non-transformed, mammalian cell is a fibroblast, a keratinocyte, a mammary gland epithelial cell, or a vascular endothelial cell.
 5. The isolated senescent cell of claim 4, wherein said juvenile, non-transformed, mammalian cell is a fibroblast cell.
 6. The isolated senescent cell of claim 5, wherein said juvenile, non-transformed, mammalian cell is a human fibroblast cell.
 7. The isolated senescent cell of claim 1, wherein said juvenile, non-transformed, mammalian cell is a cell of 0 to 50 population doublings (PD).
 8. The isolated senescent cell of claim 1, wherein said protein that directly phosphorylates p38 protein is selected from the group consisting of: MKK6 protein or MKK3 protein.
 9. The isolated senescent cell of claim 1, wherein said exogenous gene is introduced in said juvenile, non-transformed, mammalian cell using a retrovirus, an adenovirus, DEAE-dextran method, calcium phosphate-DNA precipitation method, transfection reagent or electroporation.
 10. A method for preparing a senescent cell, comprising: introducing and expressing an exogenous gene in a juvenile, non-transformed, mammalian cell; wherein said gene codes for a protein that directly phosphorylates p38 protein. 